PROJECT SUMMARY/ABSTRACT Recent advances in clinical gene therapy highlight the need for improved lentiviral vector (LV) and adeno- associated viral (AAV) vector manufacture and utilization efficiencies for enhanced therapeutics outcomes. Expression Therapeutics in collaboration with Emory University are developing a microfluidic (Fluidic), technology-based solution that overcomes the mass-transport and diffusion limitations of current transduction platforms to enhance ex vivo vector gene transfer kinetics and efficiency. This novel ex vivo transduction platform is flexible in design, scalable, and compatible with standard cell culture transduction reagents and vector preparations as it mechanistically relies solely on physical principles. Using hematopoietic cell lines, primary human T-cells, and primary hematopoietic stem/progenitor cells, we have demonstrated that ?Fluidic transduction occurs up to 5-fold faster and requires ~1/20th of LV needed in conventional clinical transduction protocols. In vivo application of ?Fluidics using hematopoietic stem/progenitor cells of C57BL/6J hemophilia A mice transduced with factor VIII-encoding LV and transplanted into hemophilic donors demonstrated that LV usage and transduction time is significantly reduced with our platform, without loss of hematopoietic stem cell engraftment potential. The milestones proposed for the current phase I SBIR application are to 1) design and test microfluidic devices scaled-up to process > 108 target cell for genetically-modified cell product manufacture validation at clinically-relevant scales, 2) validate these devices using clinically-relevant primary human cell targets (CD34+ cells and CD3+ T cells), and 3) explore the opportunity for application of the microfluidic transduction devices to ex vivo AAV gene transfer, which is being pursued clinically for ex vivo gene-editing and transient gene expression applications.